Aromatic compounds belong to one of the most widely distributed classes of organic compounds in nature, and a significant number of xenobiotics belong to this family of compounds. Since many habitats containing large amounts of aromatic compounds are often anoxic, the anaerobic catabolism of aromatic compounds by microorganisms becomes crucial in biogeochemical cycles and in the sustainable development of the biosphere. The mineralization of aromatic compounds by facultative or obligate anaerobic bacteria can be coupled to anaerobic respiration with a variety of electron acceptors as well as to fermentation and anoxygenic photosynthesis. Since the redox potential of the electron-accepting system dictates the degradative strategy, there is wide biochemical diversity among anaerobic aromatic degraders. However, the genetic determinants of all these processes and the mechanisms involved in their regulation are much less studied. This review focuses on the recent findings that standard molecular biology approaches together with new high-throughput technologies (e.g., genome sequencing, transcriptomics, proteomics, and metagenomics) have provided regarding the genetics, regulation, ecophysiology, and evolution of anaerobic aromatic degradation pathways. These studies revealed that the anaerobic catabolism of aromatic compounds is more diverse and widespread than previously thought, and the complex metabolic and stress programs associated with the use of aromatic compounds under anaerobic conditions are starting to be unraveled. Anaerobic biotransformation processes based on unprecedented enzymes and pathways with novel metabolic capabilities, as well as the design of novel regulatory circuits and catabolic networks of great biotechnological potential in synthetic biology, are now feasible to approach.

Anaerobic Catabolism of Aromatic Compounds: a Genetic and Genomic View

Bacterial expression systems for recombinant protein production: E. coli and beyond.

The provision of pure enantiomers is of increasing importance not only for the pharmaceutical industry but also for agrochemistry and biotechnology. In general, there are two rival approaches to provide pure enantiomers. The "chiral" approach is based on developing an asymmetric synthesis of just one of the enantiomers, while the "racemic" approach is based on separating mixtures of the two enantiomers. In the last few years remarkable progress has been achieved in the latter area. This Review focuses in particular on enantioselective crystallization processes and preparative chromatography, including hybrid processes and the incorporation of racemization steps. Several examples from our research are used for illustration purposes.

/pdf/processes-to-separate-enantiomers-2o17sxes0g.pdf

Processes to separate enantiomers.

Processes to separate enantiomers

Widespread pollution of terrestrial ecosystems with petroleum hydrocarbons (PHCs) has generated a need for remediation and, given that many PHCs are biodegradable, bio- and phyto-remediation are often viable approaches for active and passive remediation. This review focuses on phytoremediation with particular interest on the interactions between and use of plant-associated bacteria to restore PHC polluted sites. Plant-associated bacteria include endophytic, phyllospheric, and rhizospheric bacteria, and cooperation between these bacteria and their host plants allows for greater plant survivability and treatment outcomes in contaminated sites. Bacterially driven PHC bioremediation is attributed to the presence of diverse suites of metabolic genes for aliphatic and aromatic hydrocarbons, along with a broader suite of physiological properties including biosurfactant production, biofilm formation, chemotaxis to hydrocarbons, and flexibility in cell-surface hydrophobicity. In soils impacted by PHC contamination, microbial bioremediation generally relies on the addition of high-energy electron acceptors (e.g., oxygen) and fertilization to supply limiting nutrients (e.g., nitrogen, phosphorous, potassium) in the face of excess PHC carbon. As an alternative, the addition of plants can greatly improve bioremediation rates and outcomes as plants provide microbial habitats, improve soil porosity (thereby increasing mass transfer of substrates and electron acceptors), and exchange limiting nutrients with their microbial counterparts. In return, plant-associated microorganisms improve plant growth by reducing soil toxicity through contaminant removal, producing plant growth promoting metabolites, liberating sequestered plant nutrients from soil, fixing nitrogen, and more generally establishing the foundations of soil nutrient cycling. In a practical and applied sense, the collective action of plants and their associated microorganisms is advantageous for remediation of PHC contaminated soil in terms of overall cost and success rates for in situ implementation in a diversity of environments. Mechanistically, there remain biological unknowns that present challenges for applying bio- and phyto-remediation technologies without having a deep prior understanding of individual target sites. In this review, evidence from traditional and modern omics technologies is discussed to provide a framework for plant-microbe interactions during PHC remediation. The potential for integrating multiple molecular and computational techniques to evaluate linkages between microbial communities, plant communities and ecosystem processes is explored with an eye on improving phytoremediation of PHC contaminated sites.

/pdf/the-interaction-between-plants-and-bacteria-in-the-2lq1v88m25.pdf

The Interaction between Plants and Bacteria in the Remediation of Petroleum Hydrocarbons: An Environmental Perspective

In an effort to identify alternate recombinant gene expression systems in Pseudomonas fluorescens, we identified genes encoding two native metabolic pathways that were inducible with inexpensive compounds: the anthranilate operon (antABC) and the benzoate operon (benABCD). The antABC and benABCD operons were identified by homology to the Acinetobacter sp. anthranilate operon and Pseudomonas putida benzoate operon, and were confirmed to be regulated by anthranilate or benzoate, respectively. Fusions of the putative promoter regions to the E. coli lacZ gene were constructed to confirm inducible gene expression. Each operon was found to be controlled by an AraC family transcriptional activator, located immediately upstream of the first structural gene in each respective operon (antR or benR). We have found the anthranilate and benzoate promoters to be useful for tightly controlling recombinant gene expression at both small (< 1 L) and large (20 L) fermentation scales.

/pdf/identification-of-anthranilate-and-benzoate-metabolic-2fnkzz0lsj.pdf

Identification of anthranilate and benzoate metabolic operons of Pseudomonas fluorescens and functional characterization of their promoter regions

Aromatic ring hydroxylating dioxygenases play a key role in the biodegradation of numerous environmental pollutants, both in the natural environment (via natural attenuation) and in the engineered bioremediation systems. Recent structural and mechanistic information, together with enzyme engineering and strain construction strategies should allow the development of engineered microorganisms with new and/or optimized degradation abilities. The continued application of these approaches should also facilitate the development of Rieske non-heme iron dioxygenases with requisite selectivities for specific opportunities in target direct biocatalysis or metabolic engineering.

Aromatic Ring Hydroxylating Dioxygenases

Compositions and methods for improving expression and/or secretion of protein or polypeptide of interest in a host cell are provided. Compositions comprising a coding sequence for a bacterial secretion signal peptide are provided. The coding sequences can be used in vector constructs or expression systems for transformation and expression of a protein or polypeptide of interest in a host cell. The compositions of the invention are useful for increasing accumulation of properly processed proteins in the periplasmic space of a host cell, or for increasing secretion of properly processed proteins from the host cell. In particular, the secretion signal peptides comprise a protein disulfide isomerase C (dsbC), a mutant phosphate binding protein (pbp*), a protein disulfide isomerase A (dsbA), a CupA2, a CupB2,. a CupC2, a NikA, a FIgI, a tetratricopeptide repeat family protein (ORF5550), a toluene tolerance protein (Ttg2C), or a methyl accepting chemotaxis protein (ORF8124) secretion signal.

Bacterial leader sequences for increased expression

Since the initial discovery of toluene dioxygenase (TDO) by David Gibson and coworkers (Yeh et al. 1977; Subramanian et al. 1979), aromatic hydrocarbon dioxygenases have been reported to catalyze the initial reaction in the bacterial biodegradation of a diverse array of aromatic and polyaromatic hydrocarbons, aromatic acids, chlorinated aromatic, and heterocyclic aromatic compounds. To date, more than 100 aromatic compound dioxygenases have been described in the literature based on biological activity or nucleotide sequence identity. These enzymes are cofactor-requiring multicomponent heteromultimeric proteins (EC 1.14.12) that catalyze the initial activation through reductive dihydroxylation of their substrates, and are distinct from aromatic ring-cleavage (or ring-fission) dioxygenases (EC 1.13.11), which act on the catechol intermediates in many of the same catabolic pathways. This chapter will focus primarily on the aromatic ringhydroxylating dioxygenases (Rieske non-heme iron dioxygenases; EC 1.14.12), which initiate an attack on aromatic hydrocarbons, heterocycles and related compounds carrying various substituents (Cl, NO2). The aspects described will relate to dioxygenase discovery, classification, enzymology, structure, electron transport, mechanism and applications.

Sol M. Resnick

Papers

Identification of anthranilate and benzoate metabolic operons of Pseudomonas fluorescens and functional characterization of their promoter regions

Aromatic Ring Hydroxylating Dioxygenases

Bacterial leader sequences for increased expression

Aromatic Hydrocarbon Dioxygenases

Soluble periplasmic production of human granulocyte colony-stimulating factor (G-CSF) in Pseudomonas fluorescens.